(none)

Jeff Kramer jeff at COMPBIO.MED.WAYNE.EDU
Tue Aug 1 16:57:44 EST 1995


Mike Holloway wrote:
--------------------------------------------------------------------------------
I'm giving up on the pET system and wondered if anyone else has had 
similar experiences.
	...stuff deleted...
Anyone had experience with pET vectors?
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I have spentf years of my life trying to get lipoxygenase expression using
pET 3a while a grad student working on my PhD.  Even after I got expression, I
rarely got anywhere near reproducible amounts of protein.  The specific activity
was, thankfully, pretty reproducible.  By "pretty" I mean with in ~15%,
which was less than I would have liked, but good enough for my purposes.  What
it meant was that I did more repeat experiments than I probably would have had
to otherwise, to make sure my data was statistically significant.  Again, I was
fortunate in that three of the four site directed mutants that I made were dead
as door nails, and the forth hovered right around 50%, so even with the varia-
tion in specific activity, I could still say some meaningful things about lipoxy
genase.  Still, I wish I had chosen a different system.  I've even humbly sub-
mitted to my former boss that getting lox expression from yeast would make for
a nice Masters project for someone, and wouls save the entire lab a heck of a
lot of headache.  So in short, my advice is, give up on the pET vectors.  I
believe NEB now sells them, and I love NEB (no affiliation, etc).  I have never
gotten anything from them that wasn't excellant (I got the pET 3a before NEB
liscenced the system), but I wouldn't encourage anyone to use pET vectors, if
another system is readily available...

							-Jeff

					Jeffrey A. Kramer, Ph.D.
					Wayne State University Medical Center
					C.S. Mott Center for Human Growth and
						Development
					275 E. Hancock
					Detroit, MI  48201
					jeff at compbio.med.wayne.edu
					http://compbio.med.wayne.edu/~jeff/
 



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